Interaction of polymorphonuclear leukocytes and viruses in humans: adherence of polymorphonuclear leukocytes to respiratory syncytial virus-infected cells

The nature of neutrophil-respiratory syncytial virus (RSV) interaction was investigated by assessing factors that influence neutrophil adherence to RSV-infected tissue culture monolayers. The adherence of neutrophils to infected cells was directly proportional to the degree of RSV replication as evidenced by infectious virus production, cytopathological changes, or viral antigen appearance. Sixty-one percent of the neutrophils adhered to the RSV-infected cells as compared with 52.7% on noninfected monolayers (P less than 0.05). The addition of RSV-specific antibody markedly increased polymorphonuclear leukocyte adherence to 88.5% (P less than 0.001). Complement in the absence of antibody augmented polymorphonuclear leukocyte adherence, but to a lesser degree, 69.0% (P less than 0.025). Arachidonic acid metabolism appeared to play a critical role in the adherence process; thromboxane was the single most important arachidonic acid metabolite. Inhibition of thromboxane synthesis reduced antibody-dependent polymorphonuclear leukocyte adherence on RSV-infected cells to 52.3% (P less than 0.025). These observations suggest a role for neutrophils in RSV infection. It is proposed that neutrophils may participate in RSV infection at the site of viral replication through the attachment to infected cells and the subsequent release of mediators of inflammation.     

Interaction of polymorphonuclear leukocytes, bradykinin and carragenin. Transference reaction in the rat

1. Injected in the paw of the rat, polymorphonuclear leucocytes do not increase the oedematogen action of bradykinin, but increase the action of lambda carrageenan. 2. This potentiation of carrageenan action is not modified when PG biosynthesis in leucocytes is inhibited by indomethacin or aspirin. It does not appear in rats previously treated by indomethacin or aspirin. 3. Our results suggest that rat polymorphonuclear leucocytes increase the inflammatory reaction, when they are stimulated by carrageenan, by the release of a phospholipase A2 activity which induces PG biosynthesis in rat paw tissues.     

The role of adhesive interactions and extracellular matrix fibronectin from human polymorphonuclear leukocytes in the respiratory burst

The Arg-Gly-Asp (RGD) tripeptide and ajoene were used for studying the role of adhesive receptors in the respiratory burst. Activation of the respiratory burst was examined by using luminol-dependent and lucigenin-dependent chemiluminescence. Recently, it was shown that ajoene, (E, Z)-4,5,9-trithiadodeca-1,6,11-trien-9-oxide, a substance isolated from garlic extract, inhibits the binding of fibrinogen to activated platelets by direct interaction with fibrinogen receptor (Apitz-Castro, R., Lederma, E., Escalante, J. and Jain, M.K. (1986) Biochem. Biophys. Res. Commun. 141, 145-150). Taking into consideration the structural and functional similarity of integrins, it would be reasonable to assume that ajoene as well as RGD can inhibit adhesive interactions of human neutrophils. We have shown that the effect of various activators on the respiratory burst was abolished by ajoene or RGD treatment. The inhibitory effect of RGD and ajoene was dose-dependent. The treatment of neutrophils with antiserum against human plasma fibronectin inhibited the respiratory burst in response to formyl-methionyl-leucylphenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). This effect is dose-dependent and reversible with the addition of fibronectin. These data indicate that the respiratory burst in human neutrophils is mediated by the integrin family of receptors and that interactions between the extracellular matrix fibronectin and cells are necessary for the respiratory burst.     

Interaction of heparin with fibronectin and isolated fibronectin domains.

Fluorescence polarization, gel exclusion chromatography and affinity chromatography were used to characterize the interaction of heparins of different size with human plasma fibronectin (Fn) and several of its isolated domains. The fluid-phase interaction of Fn with heparin was dominated by the 30 kDa and 40 kDa Hep-2 domains located near the C-terminal ends of the A and B chains respectively. The 30 kDa Hep-2A domain from the heavy chain was indistinguishable from the 40 kDa Hep-2B domain in this respect; the presence of an additional type III homology unit in the latter had no effect on the binding. Evidence was provided that each Hep-2 domain has two binding sites for heparin. The N-terminal Hep-1 domain reacted weakly in fluid phase even though it binds strongly to immobilized heparin. Fn and Hep-2 fragments were rather undiscriminating in their reaction with fluoresceinamine-labelled heparins of different sizes. However, oligosaccharides smaller than the tetradecasaccharide (14-mer) bound Fn with a 5-10-fold lower affinity. These results suggest that the Hep-2 domains of Fn are able to recognize a broad spectrum of oligosaccharides that presumably vary significantly with respect to the amount and spatial distribution of charge.     

Association of microfilament bundles with lysosomes in polymorphonuclear leukocytes

The juxtaposition of microfilament bundles and lysosomes seen both in thin-sectioned cells in the transmission electron microscope and in cryofractured cells in the scanning electron microscope, and the presence of short filamentous structures between lysosomes and microfilament bundles, suggest that microfilaments may be attached to lysosomal membranes and that these filaments may be involved in lysosomal movements. Further work is in progress to test these hypotheses.     

Interaction of chemotactic factors with human polymorphonuclear leukocytes: Studies using a membrane potential-sensitive cyanine dye

Summary Changes in the fluorescence intensity of the dye 3-3 dipentyloxacarbocyanine were measured in suspensions of purified human peripheral blood polymorphonuclear leukocytes (PMNs) during exposure to the chemotactic factors N-formyl-methionylleucyl-phenylalanine (f-met-leu-phe) and partially purified C5a. Incubation of PMNs with dye resulted in a stable fluorescence reflecting the resting membrane potential of the cell. Exposure of PMNs to dye did not affect stimulated chemotaxis or secretion. The mechanism of cell-associated dye fluorescence involved solvent effects from partitioning of the dye between the aqueous incubation medium and the cell and not dye aggregation, Chemotactically active concentrations of f-met-leu-phe (5×10^–9 m or greater) produced a biphasic response characterized as a decrease followed by an increase in fluorescence. No fluorescence response was seen in lysed PMNs, and no response was elicited by an inhibitor of f-met-leu-phe binding (carbobenzoxy-phenylalanyl-methionine). The ability of several other synthetic peptides to elicit a fluorescence response corresponded to their effectiveness as chemotactic agents. Although the first component of the response suggested a depolarization, it was not influenced by variation in the external concentration of sodium, potassium, chloride, or calcium, and could not be characterized as a membrane potential change. The second component of the response, which was inhibited by both Mg²⁺ (10mm)-EGTA (10mm) and high external potassium, was compatible with a membrane hyperpolarization. The data indicate that chemotactic factors produce changes in dye fluorescence which can, at least in part, be attributed to a hyperpolarizing membrane potential change occurring across the plasma membrane.Presented in part at the 17th Annual Cell Biology Meeting.Cell Biol. 75:103a, 1977.     

Cytochalasin binding in lymphocytes and polymorphonuclear leukocytes

The binding of [³H]cytochalasin B (CB) to intact cells was compared in lymphocytes, polymorphonuclear leukocytes (PMNLs) and erythrocytes over a broad range of cytochalasin concentrations. Binding curves consistent with the presence of high and low affinity binding sites were demonstrated in all three cell types. However, in contrast to observations in erythrocytes, in lymphocytes and PMNLs CB binding was unaffected by d-glucose. p-Hydroxymercuribenzoate and p-hydroxymercurisulfonate were only partially inhibitory and unlabeled cytochalasins E, D and A (CE, CD, CA) inhibited [³H]CB binding more effectively than unlabeled CB. While attempts to demonstrate that plasma membrane-rich subcellular fractions from lymphocytes selectively bind [³H]CB were inconclusive, radioautographic studies on unbroken cells indicated that most or all of the high affinity CB-binding sites in lymphocytes and PMNLs were in close proximity to the cell surface.     

Motility of polymorphonuclear leukocytes in malnutrition]

Random polymorphonuclear granulocytes motility has been studied in malnourished children, aged between one month and one year. A technique, which allowed to observe trajectories of individual cells has been applied. An analysis included the measurements of cells centres position. Results have indicated, that the average cell velocity (one-minute translocation) does not differ in malnutrition from that seen during convalescence period. However, distribution of cell translocation values have been statistically significantly different.     

Proton release associated with respiratory burst of polymorphonuclear leukocytes

The stimulation of polymorphonuclear leukocytes (PMNs) by phorbol-12-myristate-13-acetate in the presence of sodium fluoride caused the release of protons into the reaction medium concomitant with the generation of superoxide anions. The rates of oxygen consumption and proton release due to the metabolic burst were 16.3 +/- 3.5 and 10.2 +/- 1.1 nmol/min/10(7) cells respectively. When the superoxide anions were trapped with cytochrome c, the proton release was increased (35.8 +/- 0.5 nmol/min/10(7) cells) until the cytochrome c was reduced. Since the protons released from the activated cells would be consumed by the generated superoxide anions in the extracellular medium, the net amount of the protons released was 3-4-fold greater than that observed in the absence of extracellular cytochrome c. The increased proton release may be coupled to increased cellular respiration, since the inhibition of the respiratory burst with deoxyglucose, p-chloromercuribenzoic acid or chlorpromazine decreased the proton release. Amiloride (2 mM) inhibited the proton release by up to 40%. These observations suggest that some mechanisms other than a Na+/H+ antiport and carbon dioxide diffusion could be transporting the H+ generated in the cytosol of the activated PMNs.